Stabilization of nucleic acids in urine

ABSTRACT

A composition and method for preserving a urine sample and a preservative delivery vessel are disclosed wherein treatment of the urine sample aids in preserving circulating cell-free nucleic acids in urine over a wide range of dilution ratios within temperature fluctuations that can occur during urine sample handling, storage and transportation. The urine sample preservation composition and method and preservative delivery vessel provide a method for obtaining high quality stabilized urinary cell-free nucleic acids for clinical diagnostics development and application.

TECHNICAL FIELD

The present teachings relate to a urine preservative composition forstabilization of cell-free nucleic acid in a urine sample, a method forpreserving a urine sample and a preservative delivery vessel.

BACKGROUND

Detection of circulating cell-free DNA (cfDNA) in blood plasma derivedfrom tumor, fetus and transplanted organs has been well documented. SeeU.S. Patent Applications 2014/0080112 and 2010/0184069 incorporated byreference herein. These circulating cfDNAs can pass from the bloodthrough the kidney barrier into urine. The obvious advantages andnon-invasive nature of urine sampling makes urine a useful source offetal and tumor DNA for the development of noninvasive prenatal andcancer diagnostic and prognostic tests, in addition to detection andidentification if other disease states. However, the inherentinstability of cfDNA in urine hinders its clinical utility. Nucleatedcells in urine can also release genomic nucleic acids into urine leadingto an increased nucleic acid background during sample processing andstorage.

The discovery of cell-free nucleic acids in the circulation has openedup new opportunities for non-invasive diagnostic applications in cancertesting and prenatal diagnosis (Tong Y K and Lo Y M D. Diagnosticdevelopments involving cell-free (circulating) nucleic acids. ClinicaChimica Acta 2006; 363:187-196). Since the confirmation of cfDNApresence in urine, there has been much interest in the potential utilityof urinary DNA for clinical diagnostic development. (Botezatu I, SerdyukO, Potapova G, et al. Genetic analysis of DNA excreted in urine: a newapproach for detecting specific genomic DNA sequences from cells dyingin an organism. Clinical Chemistry 2000; 46:1078-1084.) The mainadvantage of urine over other body fluids (e.g., blood) is that urinesampling is truly non-invasive and it can be obtained safely and inlarge amounts with very limited training. When employing urinary cfDNA,however, it is important to minimize release of cellular DNA fromnucleated cells and stabilize cfDNA following urine collection sincecfDNA targets are present at low quantities and degrade rapidly. (Su YH, Wang M J, Aiamkitsumrit B, et al. Detection of a K-ras mutation inurine of patients with colorectal cancer. Cancer Biomarkers 2005; 1;177-182.) (Cannas A, Kalunga G, Green C, et al. Implications of StoringUrinary DNA from Different Populations for Molecular Analyses. PLoS ONE2009; 4: e6985). Urine conditions (i.e. pH, nuclease activity) aremarkedly different from those of blood. On average nuclease activity infresh urine is greater than that in blood plasma. (Cherepanova A,Tamkovich S, Pyshnyi D, et al. Immunochemical assay fordeoxyribonuclease activity in body fluids. Journal of immunologicalmethods. 2007 Aug. 31;325(1):96-103.) All articles are incorporated byreference herein.

Effective stabilization of the nucleated blood cells in urine alone isnot sufficient to effectively stabilize the native cell free DNAcompliment in fresh urine. Inhibition of the significantly highernuclease activity in urine as compared to blood plasma is critical tothe effective stabilization. Therefore, requirements for effectivestabilization of the native cell free DNA in urine samples aremulti-fold.

Effective stabilization of cell free-DNA in urine requires stabilizationof nucleated blood cells in urine, quenching of damaging freeformaldehyde that may be present from stabilizing/preserving agents andblocking of the high nuclease activity in urine. There is a need fornucleated blood cell stabilization to reduce the level of contaminatinggenomic DNA to the overall DNA yield. There is a need for apreserving/stabilizing agent which does not compromise or destroy thestructural integrity of the actionable mutations in the cell free DNAand or ctDNA due to free formaldehyde. There is a need for blockingenzymatic active (i.e. DNase activity) in fresh urine to prevent theenzymatic activity that will rapidly hydrolyze nucleic acids. Thus,there is a need for a preservative reagent that can preserve theoriginal proportion and integrity of cfDNA in urine post specimencollection.

It is also necessary to address pre-analytical issues that arise duringthe time between urine collection and cfDNA isolation. These includedelays in urine processing and specimen storage temperature. Suchconditions may cause cellular DNA contamination and subsequently altercfDNA levels circulating in urine. Thus, in order to obtain reproducibleresults, it is essential to standardize the pre-analytical procedure forurine sample handling. cfDNA preservation and stabilization in urineshould be an integral part of the non-invasive diagnostic testdevelopment using urine as the source of genetic material. Thus, thereis a need for a urine preservation method and preservation deliveryvessel that can maintain the cfDNA concentration in urine post specimencollection. There is a need for a urine preservation method andpreservation delivery vessel that offers the benefit of preserving urinecellular material that may later be processed and the nucleic acidsisolated for molecular diagnostic analysis.

SUMMARY OF THE INVENTION

The present teachings contemplate a urine preservative composition forstabilization of cell-free DNA in a urine sample. The present teachingsprovide for stabilization of nucleated blood cells in urine. The presentteachings further provide for quenching substantially all available freeformaldehyde that may be present from stabilizing/preserving agentswhich release formaldehyde. The present teachings provide for blockingof nuclease activity. Thus, the present teachings provide a preservativereagent that can preserve the original proportion and structuralintegrity of cfDNA in urine post specimen collection.

The present teachings contemplate a method that can stabilize cfDNA inurine and minimize the post-sampling urinary DNA background for anextended period of time at various storage temperatures. This newmethodology provides clinical laboratories with great flexibility andconvenience in urine sample processing, handing and storage for urinarynucleic acid testing as it eliminates the necessity for immediateseparation of supernatant after urine collection andrefrigerating/freezing urine for transport.

Such a method may comprise a step of dispensing into a specimencontainer a predetermined amount of a urine preservative by removing theurine preservative from a sealed vessel. The method may also include astep of contacting the urine preservative with a urine sample. Themethod may also include the step of sealing the specimen container withthe urine sample and the urine preservative contained within it. Thestep may also include causing the sealed specimen container to beshipped to a clinical laboratory for analysis of the urine sample. Theurine sample, upon being preserved, may be capable of analysis after aperiod at least 168 hours or at least 7 days has elapsed.

There may be a urine collection step of obtaining a urine sample from apatient into a specimen container prior to the contacting step. Theurine sample may be a random specimen, a first morning specimen, amidstream clean catch specimen, a timed collection specimen, a cathetercollection specimen, or a suprapubic specimen. The urine sample may bepreserved at the collection site, for example a doctor's office. Themethod may include a step of transporting the preserved urine sample toanother site, for example a clinical laboratory at which analysis willoccur. The clinical laboratory may perform urinalysis testing on thepreserved urine sample and report the results. The preserved urinesample may be processed for isolating circulating nucleic acids. Thepreserved urine sample may be processed for isolating any circulatingtumor cells, minimal residual disease and pathogens for diagnosticanalysis.

The method may include a preservative delivery vessel for dispensinginto a specimen container a predetermined amount of a urinepreservative. The preservative delivery vessel may include a dispensingnozzle portion that includes a sealed tip that is capable of beingruptured to define a nozzle opening. The preservative delivery vesselmay include an elongated hollow barrel portion in fluid communicationwith the dispensing nozzle portion. The barrel portion may contain abodily fluid preservative composition. The barrel portion may have aninitial internal volume and a flexible wall structure that is capable ofbeing squeezed manually by a user (e.g. in the absence of any assistivetool) for reducing the initial internal volume to a smaller volume andapplying pressure to advance the preservative composition towards andthrough the nozzle opening after the sealed tip is ruptured.

The teachings herein further contemplate that the methods disclosedherein result in preserving circulating cell-free DNA in urine over awide range of dilution ratios within temperature fluctuations that canoccur during urine sample handling, storage and transportation. Theurine sample preservation method and preservative delivery vesselprovide a method for obtaining high quality stabilized urinary cell-freeDNA for clinical diagnostics development and application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the results of unpreserved urine samples and urinesamples treated with a composition typically used for bloodstabilization including 0.17% K₃EDTA.

FIG. 2 compares the results of unpreserved urine samples and unpreservedurine samples treated with 0.9% K₃EDTA alone.

FIG. 3 compares the results of unpreserved urine samples and urinesamples treated with the urine preservative composition of the presentteachings containing at least 0.9% K₃EDTA in addition to a formaldehydescavenger and a stabilizing agent.

FIG. 4 illustrates a perspective view of the preservative deliveryvessel taught herein.

DETAILED DESCRIPTION

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the teachings, its principles,and its practical application. Those skilled in the art may adapt andapply the teachings in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present teachings as set forth are not intended as beingexhaustive or limiting of the teachings. The scope of the teachingsshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes. Other combinations are also possible as willbe gleaned from the following claims, which are also hereby incorporatedby reference into this written description.

This application claims the benefit of the filing date of U.S.Provisional Application No. 62/128,774, filed Mar. 5, 2015. The entiretyof the contents of that application are hereby incorporated by referencefor all purposes.

The urine preservative composition provides for stabilization ofcell-free nucleic acid in a urine sample. The urine sample includesnucleic acids which may be include one or more of cell free nucleicacids, circulating tumor nucleic acids, cellular nucleic acids, DNA,RNA, microRNA (miRNA), or messenger RNA (mRNA). The urine preservativecomposition may include one or more stabilizing/preserving agents. Theone or more stabilizing/preserving agents may be formaldehyde releasingagents. The urine preservative composition may include one or moreformaldehyde quenching agents. The urine preservative composition mayinclude one or more nuclease inhibitors. The urine preservativecomposition may comprise a stabilizing/preserving agent, a formaldehydequenching agent and a nuclease inhibitor in an amount that is greaterthan the amount required for stabilizing cell-free nucleic acid inblood. Considering the relatively minor in nuclease activity in urine,it is surprising that such a significant increase in nuclease inhibitor(as compared to blood stabilizers) is required to compensate for theincrease in nuclease activity.

The method may include contacting the urine sample while within thesample specimen container with a urine preservative composition thatincludes a formaldehyde releasing agent and a quenching agent. Thequenching agent may be present in an amount sufficient to react with anyfree formaldehyde so that the free formaldehyde (e.g., all of the freeformaldehyde) reacts to form a reaction product that is inert. The urinepreservative composition may also include a nuclease inhibitor in anamount significantly greater than that utilized for blood stabilization,much more than would be expected considering the minor increase innuclease activity in urine as compared to blood.

Effective stabilization of the nucleated blood cells present in urineserves to prevent contamination of the native cell free DNA complimentpresent in fresh urine. Nucleated blood cell stabilization reduces thelevel of contaminating genomic DNA in the overall DNA yield. One of themost significant challenges with respect to the detection of actionablemutations in cell free DNA and or ctDNA is the instability of thenucleated blood cells in the urine (i.e. white blood cells) aftercollection (which instability may be increase as compared to a bloodsample). Without steps to stabilize the sample, nucleated blood cellsbreak down post blood draw, resulting in an increase in genomic DNA(gDNA). The increased gDNA serves to dilute the mutation derived DNA(the cell-free nucleic acid being sought) making it much more difficultto detect the actionable mutations and requiring that the techniques bemore sensitive than those used for mutation detection performed w/tissue analysis.

The stabilizing/preserving agent may effectively stabilize/preserve thenucleated blood cells in the urine sample. The stabilizing/preservingagent may prevent cell lysis/breakage that can occur during urine samplehandling/storage and or processing. Stabilizing/preserving agents thatmay be used include, but are not limited to diazolidinyl urea (DU),dimethylol urea, 2-bromo-2-nitropropane-1,3-diol,5-hydroxymethoxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octane and5-hydroxymethyl-1-aza-3,7-dioxabicyclo (3.3.0)octane and 5-hydroxypoly[methyleneoxy]methyl-1-aza-3,7-dioxabicyclo (3.3.0)octane, bicyclicoxazolidines (e.g. Nuosept 95), DMDM hydantoin, imidazolidinyl urea(IDU), sodium hydroxymethylglycinate, hexamethylenetetramine chloroallylchloride (Quaternium-15), biocides (such as Bioban, Preventol andGrotan), a water-soluble zinc salt or any combination thereof.

Effective stabilization also requires quenching substantially allavailable free formaldehyde that may be present fromstabilizing/preserving agents which release formaldehyde. Freeformaldehyde compromises or damages the integrity of the native DNA. Ifnot quenched it will compromise or destroy the structural integrity ofthe actionable mutations in the cell free DNA and or ctDNA. Furthermore,the quenching of formaldehyde is even more important with regard tourine. As compared to a blood sample, a urine sample contains very fewbiological components with which the formaldehyde can react with. As aresult any remaining free formaldehyde present in the sample will beable to cause damage to target nucleic acids. As a result, whereas bloodsamples only require minimal quenching of the free formaldehyde, urinesamples require that all free formaldehyde be sufficiently quenched.

The formaldehyde quenching agent quenches free formaldehyde that will infact compromise or damage the integrity of the native DNA. Formaldehydequenching agents that may be used include, but are not limited toglycine, Tris(hydroxymethyl)aminomethane (TRIS), urea, allantoin,sulfites or any combination thereof.

Stabilization of cell-free DNA in urine further requires blocking ofenzymatic active (i.e. DNase activity) in fresh urine to prevent theenzymatic activity that will rapidly hydrolyze nucleic acids. If noteffectively blocked, the nucleic acid concentration will decreasequickly as a result of the enzymatic activity that will function todestroy the physical and structural integrity of the nucleic acids. Suchenzymatic activity is higher than that found in typical blood samples,but an unexpectedly large increase (at least five times that used inblood stabilization) of nuclease inhibitor is required to compensate.

The nuclease inhibitor inhibits nuclease activity in urine. Nucleaseinhibitors that may be used include, but are not limited to ethyleneglycol tetraacetic acid (EGTA), pepstatin, K₃EDTA, phosphoramidon,leupeptin, aprotinin, bestatin, proteinase inhibitor E 64 (E-64),4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) or anycombination thereof.

The urine sample preservative composition may comprise imidazolidinylurea (IDU), diazolidinyl urea (DU,) or a combination thereof, a nucleaseinhibitor and a formaldehyde scavenging agent. The urine preservativemay also comprise imidazolidinyl urea (IDU), diazolidinyl urea (DU) or acombination thereof, glycine, and K₃EDTA. The imidazolidinyl urea (IDU)may be present in a concentration (of the preservative composition postcontact with a urine sample) of about 0.5% to about 2.0%. Theimidazolidinyl urea (IDU) may be present in a concentration of about0.20% to about 4.0%. The glycine may be present in a concentration ofabout 0.01% to about 0.2%. The glycine may be present in a concentrationof about 0.01% to about 0.4%. The glycine may be present in aconcentration of at least about 0.01%, at least about 0.05%, at leastabout 0.2%, or even at least about 0.3%. The K₃EDTA may be present in aconcentration of about 0.5% to about 2.0%. The K₃EDTA may be present ina concentration of about 0.50% to about 3.6%. The K₃EDTA may be presentin a concentration of at least about 0.5%, at least about 1%, or even atleast about 2.5%. The ratio of K₃EDTA to imidazolidinyl urea (IDU) maybe 5:2. The ratio of K₃EDTA to imidazolidinyl urea (IDU) may be fromabout 1:3 to about 3:1 (e.g., about 9:10, about 9:5 or even about 9:20).The ratio of K₃EDTA to glycine may be from about 5:1 to about 100:1(e.g., about 50:1 or even about 9:1). The urine preservative may bepresent in an amount of about 1 to about 20 percent by volume of thespecimen container. The urine preservative may be present in an amountof about 1 to about 20 percent by volume of the specimen container. Ascompared with a composition for stabilizing blood, the compositionpresented herein for stabilizing urine may have an increased amount ofnuclease inhibitor and/or an increased about of formaldehyde quenchingagent.

The urine sample may be diluted 5:1 with the preservative agent. Forexample, 4 parts urine specimen to 1 part urine preservative reagent.The urine sample may be diluted 20:1 with the preservative agent. Forexample, 19 parts urine specimen to 1 part urine preservative reagent.The urine sample may be diluted 30:1 with the preservative agent. Forexample, 29 parts urine specimen to 1 part urine preservative reagent.

The sealed vessel of urine preservative may be a single unit dose. Thesingle unit dose may hold a premeasured amount of preservative. Thesingle unit dose may be used for a preserving a single specimen sample.The single unit dose may be labeled with the amount of preservative inthe unit dose. The sealed vessel may be a non-reusable containerdesigned to hold a specific amount of preservative. [0030]The urinepreservative may be directly contacted with urine sample or the urinesample may be directly contacted with the urine preservative. Thecontacting step may take place at the collection site. Preferably thecontacting step occurs within two hours of obtaining the specimen.

The shipping temperature of the sealed specimen container may be withina range from about 6° C. to about 37° C. The sealed specimen containermay be transported without refrigeration or freezing of the preservedurine sample.

The preserved urine sample may exhibit a stability for at least about 7days post treatment. The preserved urine sample may preserve theoriginal proportion and integrity of nucleic acids in the preservedurine sample. The preserved urine sample may minimize release ofcellular nucleic acids from nucleated cells and stabilize nucleic acids.The preserved urine sample may stabilize cell-free nucleic acids inurine at 6° C., 22° C. and 37° C. for at least 7 days.

The step of analysis may be performed 1, 2, 4, or 7 days after thespecimen is obtained. The analysis of the urine sample may includeurinalysis testing. The analysis of the urine sample may includeamplification and quantification of nucleic acids using PCR. Theanalysis of the urine sample may include nucleic acid sequencingtechniques. The analysis of the urine sample may include isolatingnucleic acids from the preserved urine sample and analyzing the isolatednucleic acids to identify a pathological condition.

The urine sample method may be performed in the absence of refrigerationof the preserved sample. The urine sample method may be performed in theabsence freezing the preserved sample. The preserved sample may bestored at room temperature for a period of least 7 days.

The preservative composition may be present in an amount of about 1 mlto about 15 ml in the preservative delivery vessel. The preservativecomposition may be present in an amount of about 5 ml to 10 ml in thepreservative delivery vessel. The preservative composition may bepresent in an amount of about 3 ml to about 5 ml in the preservativedelivery vessel.

The preservative delivery vessel may be packaged in a plurality of vialstrips of single unit dosages. The plurality of vial strips may bepackaged in carton. The plurality of vial strips may be packaged as partof a diagnostic kit. The plurality of vials strips may be detachable.The plurality of vial strips may be separated from each other by tearingalong a perforation. The plurality of vial strips may be separated fromeach other by cutting along a specified marking.

The preservative delivery vessel may be formed by injection molding. Thepreservative delivery vessel may be an injection molded unit dose vial.The injection molding material may include thermoplastic orthermosetting polymers.

The effect of different urine preservative concentrations on stabilityof urinary cf DNA was examined and compared to untreated urine samples.Untreated urine samples showed a significant decrease in cell-free DNAconcentrations of both oncogene KRAS and housekeeping gene β-actin onday 4 and 7 post specimen collection at room temperature. In contrast,cell-free DNA concentrations remained stable for at least 7 days inurine samples treated 5:1 to 30:1 with urine preservative. Moreover, thepreservative reagent also stabilized cell-free DNA in urine at 6° C.,22° C. and 37° C. for up to 7 days, whereas untreated samples showed asignificant degradation of cell-free DNA at 22 ° C. and 37° C.

EXAMPLES

To study the effect of different urine preservative concentrations onthe stability of urinary cell-free DNA, treated urine samples areexamined and compared to untreated urine samples. The first-voidedmorning urine samples are diluted 5:1, 20:1 or 30:1 with thepreservative reagent and stored at room temperature with untreated urinespecimens in parallel. Aliquots are removed at specified time points.Cell-free DNA is purified from urine and quantified by a Droplet DigitalPCR (ddPCR) assay.

The first-voided morning urine collected from healthy volunteers istreated with urine preservative and is stored at specified temperatureswith untreated samples in parallel. Aliquots of urine (5 mL) are removedfrom each sample on day 0, 4 and 7, respectively. These aliquots arecentrifuged at room temperature at 4000 rpm for 10 minutes. 4 mL ofsupernatant is carefully removed without disturbing pellets andtransferred to a new tube using a pipette followed by cfDNA extraction.Urine cfDNA is purified using the commercially available QIAamp®Circulating Nucleic Acid Kit (QIAGEN®, Santa Clarita, Calif.). Foroptimal results, the manufacturer's recommended protocol is modifiedslightly by increasing the duration of the proteinase K treatment from30 min to 1 hour at 60° C. The PCR is performed using the QX100 DropletDigital PCR system (Bio-Rad, Hercules, Calif.). The KRAS copy numberassay kit is purchased from Applied Biosystems (Foster City, Calif.).Primers and the probe for the ddPCR quantification of human β-actinarepurchased from Integrated DNA Technologies (Coralville, Iowa).

Results show urinary cfDNA is stabilized by urine preservative over awide range of dilution ratio at room temperature. Urine was treated 5:1,20:1 or 30:1 with the urine preservative disclosed herein and stored atroom temperature. On day 0, 4 and 7, cfDNA is isolated from urinesamples and quantified by ddPCR. The concentration of KRAS genedecreased significantly in untreated urine on day 4 and 7, whereas itremained stable in all treated urine samples for at least 7 days postspecimen collection

To study the effect of storage temperature on cell-free DNAconcentration, both treated and untreated urine samples were stored at6° C., 22° C. and 37° C. Similarly, cell-free DNA was extracted atvarious time points and quantified by ddPCR.

The first-voided morning urine collected from healthy volunteers wastreated with urine preservative and stored at specified temperatureswith untreated samples in parallel. Aliquots of urine (5 mL) are removedfrom each sample on day 0, 4 and 7, respectively. These aliquots arecentrifuged at room temperature at 4000 rpm for 10 minutes. 4 mL ofsupernatant was carefully removed without disturbing pellets andtransferred to a new tube using a pipette followed by cfDNA extraction.Urine cfDNA is purified using the commercially available QIAamp®Circulating Nucleic Acid Kit (QIAGEN®, Santa Clarita, Calif.). Foroptimal results, the manufacturer's recommended protocol is modifiedslightly by increasing the duration of the proteinase K treatment from30 min to 1 hour at 60° C. The PCR was performed using the QX100 DropletDigital PCR system (Bio-Rad, Hercules, Calif.). The KRAS copy numberassay kitiss purchased from Applied Biosystems (Foster City, Calif.).Primers and the probe for the ddPCR quantification of human β-actin arepurchased from Integrated DNA Technologies (Coralville, Iowa).

Results show urinary cfDNA stabilized by urine preservative at variousstorage temperatures. Urine samples are treated 20:1 with urinepreservative and stored at 6° C. or 37° C., respectively. cfDNA isisolated from urine samples at specified time points and quantified byddPCR. The cfDNA (KRAS) is stabilized in all treated urine samples forat least 7 days at either 6° C. or 37° C. No statistically significantdifference in cfDNA concentration is found between stored samples andday 0 samples. Similar results are observed for the house keeping geneβ-actin.

Examples 1 and 2 demonstrate the effect of different urine preservativeconcentrations on stability of urinary cfDNA compared to untreated urinesamples. Untreated urine samples show a significant decrease in cfDNAconcentrations of both oncogene KRAS and housekeeping gene β-actin onday 4 and 7 post specimen collection at room temperature. In contrast,cfDNA concentrations remained stable for at least 7 days in urinesamples treated 5:1 to 30:1 with urine preservative. Moreover, thepreservative reagent also stabilized cfDNA in urine at 6° C. or 37° C.for up to 7 days.

In addition, the effect of different reagent formulations on stabilityof urinary cfDNA is examined and compared to untreated urine samples.The urine samples are diluted with various reagent formulations andstored at room temperature with untreated urine specimens in parallel.Aliquots are removed at specified time points. cfDNA (β-actin) ispurified from urine and quantified by a Droplet Digital PCR (ddPCR)assay.

FIG. 1 compares the results of unpreserved urine samples and urinesamples treated with a reagent for stabilizing blood including about 300to about 700 g/L IDU, from about 60 to about 100 g/L K₃EDTA, and about20 to about 60 g/L glycine. Results show the reagent for stabilizingblood does not effectively block urine nuclease activity, resulting inDNA degradation. A decrease in cfDNA is associated with nucleaseactivity and an increase with genomic DNA (unstable WBC). Resultsindicate that the reagent for stabilizing blood fails to stabilize cfDNAin urine.

FIG. 2 compares the results of unpreserved urine samples and unpreservedurine samples treated with 0.9% K₃EDTA. The treated urine samples weretreated with a K₃EDTA concentration at around 5× the concentrationcontained in a commercial K₃EDTA blood collection tube (i.e. 0.17% vs.0.9%). The nuclease activity was completely blocked by increasing theconcentration of the nuclease inhibitor. Results indicate treatment with0.9% K₃EDTA prevents degradation of the native cell free DNA complimentin urine. However, cellular DNA is released in the absence of aneffective nucleated blood cell stabilizer (i.e. WBC degradation).

Urine conditions are markedly different from those of blood. Fresh urinehas a higher DNase activity as compared to blood plasma. As seen in FIG.1, in the absence of nuclease inhibition the native cell free DNApresent in fresh urine is grossly unstable, regardless of thepresence/concentration of the fixative/stabilizer as commonly used inblood stabilization. FIGS. 1 and 2 illustrate the marked need for aheightened level of nuclease activity inhibition.

FIG. 3 compares the results of unpreserved urine samples and urinesamples treated with the urine preservative composition disclosedherein. The treated urine samples were treated with a urine preservativecomposition including about 300 to about 700 g/L imidazolidinyl urea(IDU), about 0.9% K₃EDTA (e.g., from about 100g/L to about 300 g/L) andat least about 30g/L glycine. The results indicate that the urinepreservative composition stabilizes cfDNA in urine. Therefore, thepresent teachings provide a preservative reagent that can preserve theoriginal proportion and integrity of cfDNA in urine post specimencollection.

FIG. 4 shows an example container for carrying the urine preservativecomposition in accordance with the teachings herein. Each individualcontainer can be separately opened and added to a urine sample inaccordance with the teachings herein.

The explanations and illustrations presented herein are intended toacquaint others skilled in the art with the invention, its principles,and its practical application. Those skilled in the art may adapt andapply the invention in its numerous forms, as may be best suited to therequirements of a particular use. Accordingly, the specific embodimentsof the present invention as set forth are not intended as beingexhaustive or limiting of the invention. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but should instead be determined with reference to theappended claims, along with the full scope of equivalents to which suchclaims are entitled. The disclosures of all articles and references,including patent applications and publications, are incorporated byreference for all purposes. Other combinations are also possible as willbe gleaned from the following claims, which are also hereby incorporatedby reference into this written description.

1: A method for preserving a urine sample comprising the steps of: (a)dispensing into a specimen container a predetermined amount of a urinepreservative by removing the urine preservative from a sealed vessel;(b) contacting the urine preservative with a urine sample; (c) sealingthe specimen container with the urine sample and the urine preservativecontained within it; and (d) causing the sealed specimen container to beshipped to a clinical laboratory for analysis of the urine sample;wherein the urine sample, upon being preserved is capable of analysisafter a period at least 168 hours has elapsed. 2: The method of claim 1,wherein the urine preservative comprises: imidazolidinyl urea (IDU),diazolidinyl urea (DU) or a combination thereof, and glycine. 3: Themethod of claim 2, wherein the urine preservative includes K₃EDTA in anamount of at least five times what would be necessary for inhibition ofnuclease activity in a blood sample. 4: The method of claim 2, whereinthe urine preservative is present in an amount of about 1 to about 20percent by volume of the specimen container. 5: The method of claim 2,wherein the sealed vessel is a single unit dose. 6: The method of claim3, wherein the urine preservative is directly contacted with urinesample or the urine sample is directly contacted with the urinepreservative. 7: The method of claim 2, wherein the contacting step isperformed within about two hours of obtaining the specimen. 8: Themethod of claim 1, wherein the shipping temperature of the sealedspecimen container is within a range from about 6° C. to about 37° C. 9:The method of claim 1, wherein the sample exhibits a stability for atleast about 7 days post treatment. 10: The method of claim 1, whereinthe step of analysis is performed 1, 2, 4, or 7 days after the specimenis obtained. 11: The method of claim 10, where the analysis of the urinesample includes nucleic acids from the preserved urine sample andanalyzing the isolated nucleic acids to identify a pathologicalcharacteristic. 12: The method of claim 1, wherein the method isperformed in the absence of refrigeration. 13: The method of claim 2,wherein the urine includes one or more of cell free nucleic acids,cellular nucleic acids, DNA, circulating tumor DNA (ctDNA), RNA,microRNA (miRNA), or messenger RNA (mRNA). 14: The method of claim 2,wherein nucleic acid analysis is performed on one or more of a cell freeDNA, circulating tumor DNA (ctDNA), cell free RNA, mRNA, or miRNA fromwithin the urine. 15: The method of claim 1, wherein the sealed vesselcontains preservative composition present in an amount of about 1 toabout 15 ml. 16: The method of claim 1, wherein the sealed vessel ispackaged in a plurality of vial strips of single unit dosages. 17: Themethod of claim 1, wherein the sealed vessel is formed by injectionmolding. 18: A composition comprising: (a) a urine sample; (b) anuclease inhibitor present in a concentration of at least 0.5% of thecomposition; (c) a preservative present in a concentration of from about0.2% to about 4% of the composition; (d) a formaldehyde quencher presentin a concentration of at least 0.01%. 19: A urine preservativecomposition for maintaining structural integrity of nucleated bloodcells and cell-free nucleic acids in a urine sample comprising: (a) anuclease inhibitor present in an amount of from about 100 to about300g/L of the composition; (b) a preservative present in an amount offrom about 300 to about 700 g/L of the composition; (c) a formaldehydequencher present in an amount of at least about 30 g/L of thecomposition.